In vitro regeneration and in planta transformation protocols were standardized for the black gram (Vigna mungo L.) cv. VBN 3, an important grain legume. An efficient regeneration protocol was developed using 1-week-old de-embryonated cotyledon as explants. The maximum frequency of shoot regeneration (72.6%), multiple shoot induction (6.0 ± 0.3) and shoot elongation was achieved on MS medium supplemented with 3.0 mg/l 6-benzyl amino purine (BAP) after 4 weeks of culture. Up to 65% of the regenerated shoots were rooted on MS medium containing 0.5 mg/l α-naphthalene acetic acid (NAA) within 3 weeks of culture. The in vitro-raised plantlets were successfully hardened first under culture room conditions with 62% survival rate and then in greenhouse. A binary vector pBinAR harbouring cry1AcF gene isolated from E. coli competent cells was transformed into Agrobacterium strain EHA105 using freeze-thaw method and its presence was confirmed by colony PCR analysis of the selected single colonies for examining the presence of the npt II and cry1AcF genes. The integrity of plasmid was checked by restriction analysis. Agrobacterium tumifaciens-mediated transformation was performed using strain EHA105 harboring the binary vector pBinAR carrying cry1AcF gene under the control of CaMV35s promoter where cry1AcF as insect resistance gene and npt II gene as a selectable markers. Antibiotic sensitivity test was conducted with sprouted half-seed and 1-week-old cotyledons with kanamycin at 50 mg/l completely inhibited the shoot formation and explants survival. Genetic transformation experiments were carried out using cotyledonary explants and it was found to be unsuccessful event while regenerating the transformed cotyledons due to its recalcitrance nature. Therefore, Agrobacterium mediated in planta transformation was performed using sprouted half-seed explants of VBN 3. The transformation event consisted of sonicating the explants for 3 minutes and vacuum infiltration (750 mm of Hg) for 2 minutes in Agrobacterium (pBinAR-cry1AcF) and co-cultivation for 3 days in MS medium with acetosyringone (100µM). The transformed explants forming shoots were selected in MS medium supplemented with kanamycin 50 mg/l. The non-transformed shoots were completely bleached after selection. The presence and integration of npt II and cry1AcF transgenes into the black gram genome was confirmed by polymerase chain reaction (PCR). PCR analysis in 20 selected putative transformed black gram plantlets did not show amplification for cry1AcF and npt II genes. Work is in progress to grow the T0 seeds for molecular characterization of the inserted transgene among T1 plants.

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